Abstract
The Drosophila testis and ovary are major genetically tractable systems for studying stem cells and their regulation. This has resulted in a deep understanding of germline stem cell regulation by the microenvironment, or niche. The male and female germline niches differ. Since sex is determined through different mechanisms in the soma than in the germline, genetic or physical manipulations can be used to experimentally mismatch somatic and germline sexual identities. The phenotypic consequences of these mismatches have striking similarities to those resulting from manipulations of signals within the niche. A critical role of the germline sex determination pathway may therefore be to ensure the proper receipt and processing of signals from the niche.
1. INTRODUCTION
In Drosophila, germline stem cells (GSCs) are situated in a niche at the anterior end of the adult gonad while mature gametes are localized to the posterior, such that an anatomical axis of germ cell proliferation and differentiation is established in both sexes. The somatic cells adjacent to the stem cells constitute and provide the physical microenvironment, the stromal niche, that supports the stem cells. Stem cell niches exist in several adult tissues such as the gut epithelium, skin, neural tissues, hematopoietic system, and the germline (Ohlstein et al., 2004). Short-range signals from the niche prevent differentiation while maintaining stem cell fate. The sex-specific germline serves as a valuable system for understanding the errors that result from perturbation of this important microenvironment.
In this chapter, we outline the differences in cellular architecture between male and female gonads and discuss major signaling events that influence development of the male versus female germline. We also highlight the importance of sex-specific germline niches in influencing germline sex as can be inferred from the dramatic consequences observed when germ cells are within a sex-mismatched somatic environment.
2. SEXUALLY DIMORPHIC NICHES REGULATE GERM CELLS
Drosophila gonads are sexually dimorphic (Compare Figs. 4.1 and 4.3). Despite their stark morphological differences there are analogous mechanisms employed during gametogenesis by both sexes while crucial differences remain.
Figure 4.1.
Male germ cell development. (A) Photomicrograph of a single adult testis. (B) Diagram of the apical tip of the testis. For details see text section 2.1—the male niche. Anterior top.
Figure 4.3.
Female germ cell development. Photomicrographs of (A) a single adult ovary and (B) a single ovariole. Scale bar shown. (C) Diagram of germarium. (For details see text section 2.2—the female niche). (Image adapted from King, 1970). Anterior top.
2.1. The male niche and proliferative zone
2.1.1. Cellular architecture
The testis is a long coiled tube containing germ cells in varying stages of spermatogenesis. At the apex of the testis are the niche and the mitotic proliferative zone (reviewed in Fuller, 1993). At the core of the niche is a cluster of 10–12 small densely packed somatic cells called the hub (Hardy et al., 1979). There are 6–9 germline stem cells (GSCs) arranged around the hub in a characteristic rosette pattern. Each GSC is enclosed by a pair of somatic stem cells (SSCs) also known as cyst progenitor cells. The GSCs and the SSCs maintain close contact with the apical hub through thin cytoplasmic extensions (Fig. 4.1). GSCs divide asymmetrically to give rise to two daughter cells, one of which remains adjacent to the hub and retains its GSC identity while the other is displaced away from the hub. The daughter that exits from the niche, the gonialblast, is enveloped by two cyst cells (nondividing progeny of SSCs). Gonialblasts divide to generate spermatogonia, which undergo four rounds of synchronous mitotic divisions to generate a cohort of 16 cells. Spermatogonia undergo premeiotic DNA replication and after premeiotic S-phase, the 16 cells are referred to as primary spermatocytes. Primary spermatocytes undergo meiosis (after meiosis I, the cells are called secondary spermatocytes) to generate 64 spermatids and ultimately sperm (for details see Fuller, 1993). The asymmetry of GSC division is the key to stem cell maintenance—the cell maintaining contact with the hub remains a GSC while the gonialblast, which has no contact with the hub begins a program of cell division.
Much work has been undertaken to characterize the mechanism underlying this asymmetric division. The orientation of the mitotic spindle relative to an anchoring complex between the hub and the GSC regulates, at least in part, the asymmetric division of male GSCs (Fig. 4.2A). The mitotic spindle lies perpendicular to the hub and is set up by the positioning of the centrosomes during interphase (Yamashita et al., 2007). Upon centrosome duplication, the mother centrosome remains anchored to the hub-GSC interface and is inherited by the new GSC, while the daughter centrosome moves away from the hub and is inherited by the gonialblast. The integral centrosomal protein Centrosomin (Cnn) is required for centrosome positioning and spindle orientation (Yamashita et al., 2003). Since Cnn is required to anchor astral microtubules to the centrosome, it has been postulated that astral microtubules might link the centrosome and the cell cortex to properly orient the spindle. Indeed, in cnn partial loss-of-function mutants there is an increase in GSC number because instead of dividing asymmetrically, both daughters remain adjacent to the hub and thus maintain a GSC identity (Yamashita et al., 2003). This suggests that hub contact is a critical determinant of GSC identity.
Figure 4.2.
Male Niche. The germinal proliferation center of the male consists of a central core of somatic cells (the HUB) around which germline stem cells (GSC) are arranged radially. Each GSC is surrounded by two somatic stem cells (SSCs). Signaling cascades are shown separately for clarity as are spaces between the various cell types. (A) GSCs attach to the hub via adherens junctions (AJ). Orthogonally oriented mitotic spindles anchor the GSC cortex to the hub via APC2 and help ensure asymmetric division of the GSCs. (B) Secretion of the ligand Upd from hub cells activates JAK-STATsignaling in GSCs, which is required for their self-renewal. STAT phosphorylation leads to transcriptional activation of STAT responsive genes (shown as a black box since they are unknown). (C) Gbb and Dpp expressed in hub cells control GSC self-renewal by activation of BMP signaling, which leads to repression of bam transcription through the Mad/Med complexes. (D) EGF signaling is activated in somatic cyst cells by the ligand Spi secreted by the gonialblast (GB). Activated EGF signaling (through Ras and Raf) is required for the differentiation of GBs.
GSCs are attached to the hub cells via adherens junctions (Yamashita et al., 2005). Adenomatous Polyposis Coli 2 (APC2) is present at the interface between the hub and the GSCs where it colocalizes with Shotgun (Shg, the Drosophila E-Cadherin homolog), an adherens junction component and Armadillo (Arm, the Drosophila β-Catenin homolog) (Yamashita et al., 2003). GSCs of apc2 loss-of-function mutants have misoriented centrosomes and spindles, similar to those observed in cnn mutants and the number of GSCs around the hub is also increased (Yamashita et al., 2003). Thus, the astral microtubules that emanate from the centrosome appear to be captured by a protein complex containing APC2 and anchored to the interface of the GSC cortex with the hub in order to promote asymmetric divisions.
In addition to being anchored at the hub, GSCs and their progeny are also directly associated with SSCs and cyst cells. The SSCs that surround each GSC divide asymmetrically and concomitantly with the associated GSC to produce a pair of new SSCs as well as a pair of squamous nondividing daughters (cyst cells) that enclose the gonialblast and its progeny. During the course of spermatogenesis, the somatic cyst cells stretch around the developing cohort of germ cells to accommodate about a 20-fold increase in germ cell volume. Zero population growth (zpg) encodes a germline-specific gap-junction protein homologous to Innexin-4 (Inx4) and is localized at the interface between spermatogonia and somatic cyst cells as well as between primary spermatocytes and somatic cyst cells (Tazuke et al., 2002). Only a few germ cells (GSCs or gonialblasts) are present in zpg mutant testes, suggesting that gap junctions are required for the survival of the germ cells. zpg null mutant spermatogonia seem unable to progress in development suggesting that communication between spermatogonia and cyst cells via gap junctions is an important regulator of germ cell development (Gilboa et al., 2003; Tazuke et al., 2002).
The gonialblast undergoes four rounds of mitotic divisions with incomplete cytokinesis, generating a cluster of 16 interconnected spermatogonia (Rasmussen, 1973). During mitosis stable intercellular bridges called ring canals form at the site of incomplete cytokinesis. Ring canals connect the mitotic cells within a cyst, allowing them to share a common cytoplasm. As spermatogonia progress through mitotic divisions, the spectrosome (a spherical, spectrin-rich cytoplasmic organelle present in GSCs and gonialblasts) elongates into a rod-shaped structure called the fusome, which ultimately forms a highly branched vesicular structure interconnecting the cohort of mitotic germ cells via the ring canals (Hime et al., 1996). Spermatogonia next enter meiotic prophase I, which lasts about 3.5 days and are then designated as primary spermatocytes. Extensive transcription leading to about a 20-fold increase in size of the nuclei is characteristic of this stage. After the long meiotic prophase, most transcription is shut down and the cells complete meiosis to generate haploid gametes. Autoradiographical studies investigating gene expression indicate that there is little transcription postmeiotically in males, although the mechanism of transcription cessation is still unclear (Olivieri and Olivieri, 1965). The process of postmeiotic sperm morphogenesis from an immotile cell to a highly specialized motile one is called spermiogenesis. The transformations include chromatin condensation, cell and axoneme elongation, mitochondrial rearrangement, and sperm individualization. These postmeiotic differentiation processes have been extensively reviewed elsewhere (see Fuller, 1993; Renkawitz-Pohl et al., 2005).
2.2. Signaling pathways
Signaling between the germline and the niche plays a crucial role in the self-renewal, proliferation, and differentiation of the germline in both sexes. In the following sections, we will focus on key pathways conserved in both males and females, but employed differently in the two sexes. Janus kinase (Jak)/Signal Transducer and Activator of Transcription (STAT) signaling defines GSC fate, while Transforming Growth Factor β (TGF-β) signaling shields GSCs from precocious expression of genes that lead to proliferation. Finally, Epidermal Growth Factor Receptor (EGFR) signaling regulates encapsulation of germ cells by the somatic cyst cells, a process that ensures normal differentiation.
Stem cell self-renewal in males is controlled by the Jak/STAT signaling pathway which includes an extracellular cytokine ligand, Unpaired (Upd), a cytokine receptor, Domeless (Dome), a receptor activated kinase, Janus Kinase (Jak) encoded by the hopscotch locus (hop), and the transcription factor STAT92E (Kiger et al., 2001; Rawlings et al., 2004; Tulina and Matunis, 2001). Upd is specifically expressed in the hub cells. Upon glycosylation and subsequent secretion, it becomes tightly associated with the extracellular matrix, which limits its diffusion to a hub-proximal region (Harrison et al., 1998). Dome is on the surface of the neighboring GSCs and the Jak/STAT pathway is activated in GSCs following Upd binding to it. Hop dimerizes, and phosphorylates cytoplasmic Stat92E, which translocates to the nucleus to activate specific targets (Fig. 4.2B) (reviewed in Arbouzova and Zeidler, 2006). The activated Jak/STAT pathway confers stem cell identity (Brown et al., 2001; Kiger et al., 2001; Tulina and Matunis, 2001). Failure to activate the pathway results in loss of GSCs (Kiger et al., 2001; Tulina and Matunis, 2001). Conversely, ectopic expression of Upd in GSCs and SSCs results in a dramatic increase in the number of GSCs (and possibly gonialblasts) and failure to progress through spermatagonial divisions (Tulina and Matunis, 2001). Active STAT is detectable in GSCs in contact with the hub, but not in the gonialblast daughter cells resulting from asymmetric divisions of GSCs. This suggests that gonialblasts do not receive enough Upd to activate the Jak/STAT pathway, (although the situation may be more complex as loss of GSCs allows gonialblasts to assume GSC identity to repopulate the niche (Brawley and Matunis, 2004)). Thus, STAT signaling is highly restricted to cells abutting the hub and is essential for GSC self-renewal.
TGF-β signaling from the hub is required in GSCs and gonialblasts to repress differentiation (Fig. 4.2C). The pathway includes the extracellular ligands Glass bottom boat (Gbb) and Decapentaplegic (Dpp) and two-component receptor complexes which phosphorylate and activate a cytosolic R-Smad (receptor-regulated class of Smads), which then translocate to the nucleus to regulate gene expression (Reviewed in Raftery and Sutherland, 1999). The hub and somatic cyst cells express Gbb and Dpp, which act co-operatively to maintain GSC fate (Kawase et al., 2004). Loss of gbb activity results in a failure to renew the GSC population, resulting in a single cohort of spermatogonial cysts. While reduced gbb dose and loss of dpp function results in a similar phenotype, loss of dpp function alone does not result in this phenotype. This suggests that Gbb is critical while Dpp is an ancillary ligand (Kawase et al., 2004). An important repressive target of TGF-β/BMP signaling is bag of marbles (bam). Bam is expressed in spermatogonia and when it accumulates to high levels, spermatagonia cease mitotic amplification divisions and initiate the spermatocyte differentiation program (Gonczy et al., 1997; McKearin and Ohlstein, 1995; Schulz et al., 2004). Spermatogonia of loss-of-function bam mutant males do not cease amplification of mitosis and instead produce cysts of 32, 64, or more spermatogonia. Conversely, precocious expression of bam in GSCs and gonialblasts prevents amplification divisions leading to accumulation of single germ cells (GSCs and gonialblasts), which eventually die (Schulz et al., 2004). In gbb (but not dpp) mutant testes, GSCs inappropriately express Bam indicating that gbb is required for repressing bam transcription in GSCs. Interestingly, overexpression of dpp but not gbb leads to transcriptional repression of bam in all germ cells (Kawase et al., 2004). Gbb and (to a lesser extent) Dpp may act as short-range signals capable of blocking the spermatogonial program. In this model, spermatogonia undergoing mitotic divisions move away from the hub and therefore TGF-β signals, consequently Bam levels increase. Bam levels accumulate above a threshold leading to spermatogonia ending mitotic proliferation and entering meiosis (Shivdasani and Ingham, 2003). As a general rule, GSCs may need to be shielded from expression of any gene that is involved in differentiation, thus, repression of bam in GSCs might play an important permissive role in maintaining GSC identity.
EGFR signaling from the germ cells is required for proper encapsulation of gonialblasts by somatic cyst cells (Fig. 4.2D) (Schulz et al., 2002). The isolated microenvironment around the gonialblasts and spermatogonia provided by the surrounding pairs of cyst cells may help regulate the differentiation of the enclosed germ cells since EGF signaling is necessary for the spermatogonium to spermatocyte transition. EGFR is a tyrosine kinase receptor, which activates the mitogen activated protein kinase (MAPK) cascade by regulating the activities of the monomeric GTPase Raf and the serine-threonine protein kinase Ras (reviewed in Freeman, 2002). Germ cells signal via the ligand Spitz (Spi) to the surrounding cyst cells (Sarkar et al., 2007). Spi is expressed as an inactive transmembrane protein and is cleaved into a functional soluble fragment (Wasserman and Freeman, 1998). Proteolytic cleavage and activation of Spi requires the proteins Rhomboid-1 (Rho-1) and Star (Bier et al., 1990; Kolodkin et al., 1994). An additional Rhomboid protein (Rho-2) encoded by stem cell tumor (stet) is expressed in adult testes and is required in germ cells for their association with somatic cyst cells as well as for normal differentiation of germ cells (Guichard et al., 2000; Schulz et al., 2002; Wasserman et al., 2000). Star (Star) is transcribed at high levels in germ cells at the apical tip where GSCs, gonialblasts, and spermatogonia reside (Schulz et al., 2002). Genetic mosaic analyses have shown that the functions of EGFR and its downstream effector Raf are required in somatic cyst progenitor cells and somatic cyst cells but not in germ cells (Kiger et al., 2000; Tran et al., 2000). The current working model posits that Stet and Star activate Spi in germ cells, which subsequently binds to EGFR on somatic cyst cells (Schulz et al., 2002). This is supported by the presence of activated MAPK in cyst cells (Kiger et al., 2000). In stet loss-of-function testis MAPK expression is restricted to the hub cells and cyst progenitor cells, as a result mitotically active spermatogonia are interconnected by short branched fusomes suggesting failure to complete the full four rounds of mitosis (Schulz et al., 2002). There are fewer somatic cyst cells in stet mutant testis when compared to wild-type and they are round instead of bean shaped, indicating a failure to envelop the gonialblasts and subsequent spermatogonial cysts (Schulz et al., 2002). A similar phenotype is observed in Egfr, spi, and raf loss-of-function testis where GSCs, gonialblasts, and early spermatogonia accumulate (Kiger et al., 2000; Sarkar et al., 2007; Tran et al., 2000). Interestingly, in addition to accumulation of early spermatogonia, there are also spermatogonia that undergo excessive mitotic divisions. Thus, EGFR activity in somatic cyst cells is not only required to restrict self-renewal or proliferation of germ cells, but also to permit differentiation of stem cells to gonialblasts and the transition from the spermatogonia to the spermatocyte stage.
2.3. The female niche and proliferative zone
2.3.1. Cellular architecture
An ovary is subdivided into about 18 individual ovarioles, each of which functions as an independent assembly line to produce eggs (Fig. 4.3) (for details see Spradling, 1993). The germarium is located at the anterior tip of each ovariole and consists of the niche and the proliferative germ cells (Fig. 4.4C) (Brown and King, 1962; Wieschaus and Szabad, 1979). The ovarian niche is at the anterior tip of each germarium and is composed of a stack of 8–10 disc-like somatic cells, called the terminal filament, and 5–7 squamous epithelial cells, the cap cells that literally cap the underlying 2–3 GSCs. The GSCs divide asymmetrically such that the anteriorly positioned daughter cell remains in contact with the cap cells and maintains GSC identity, while the posteriorly displaced daughter cell leaves the niche, and differentiates into a cystoblast (Lin and Spradling, 1995). Interspersed between the GSCs are 4–6 escort stem cells. Escort stem cells are akin to the SSCs of males; they are interspersed between the GSCs and surround them while maintaining contact with the niche (Decotto and Spradling, 2005). The differentiated daughters of the escort stem cells, called escort cells, are displaced away from the niche after cell division and enclose the cystoblast destined for differentiation. Like their male counterparts, female cystoblasts undergo four rounds of synchronous divisions with incomplete cytokinesis to give rise to 16 cystocytes. Unlike in males where cyst cells remain in contact with the spermatocytes, in females after the 16-cell cyst forms the associated escort cells undergo apoptosis (Decotto and Spradling, 2005). The germ cell cyst then becomes tightly associated with another somatic cell type—the somatic prefollicle cells derived from two follicle stem cells maintained in two stroma-free niches at the region 2a/2b boundary (Nystul and Spradling, 2007). If there are insufficient prefollicle cells to replace the escort cells the apoptotic germline cyst progression checkpoint is activated and the germline cyst itself undergoes apoptosis (Smith et al., 2002). This suggests that female cystocyte viability requires envelopment by either escort or follicle cells, or a sequential envelopment by the two somatic cell types. Thus, while the gross cellular anatomy of the apex and the germarium are different, many principles of germarial organization are quite analogous to the testis apex.
Figure 4.4.
Female niche. The apex of an ovariole consists of terminal filament cells and cap cells which constitute the niche. GSCs are surrounded by escort stem cells (ESCs). Signaling cascades are shown separately for clarity as are spaces between the various cell types. (A) GSCs attach to the niche cells via adherens junctions (AJ). Orthogonally oriented mitotic spindles anchor the GSC cortex to the cap cells by the spectrosome. Yb, Piwi, Hh, Gbb and Dpp are expressed in the niche and act to control GSC self-renewal extrinsically. Piwi is also expressed in the GSCs and controls GSC division intrinsically. (B) Dpp/Gbbsignaling leads to repression of bam in GSCs through Mad/Med complexes. The JAK-STAT pathway is activated in ESCs and controls their maintenance. (C) Bam is expressed in cystoblasts (CBs) and is required for their differentiation.
Stem cell divisions are asymmetric by definition. This asymmetry depends on mitotic spindle orientation perpendicular to the interface of the GSCs with the niche (Deng and Lin, 1997; Yamashita et al., 2003). The spectrosome in female GSCs is localized to the apical interface with the cap cells and serves as an anchor for one of the spindle poles (Fig. 4.4A). This is in contrast to the situation in males, where the spectrosome is unanchored and does not appear to play a role in establishing spindle orientation. The female spectrosome is also closely associated with the centrosome, however, the centrosome is not required for spindle orientation (or later oogenesis) (Lin et al., 1994; Stevens et al., 2007). Thus, the asymmetric divisions of GSCs within the female niche are very reminiscent of the process in males, although there are clear differences in the mechanisms used to accomplish the task.
Both male and female GSCs are anchored to the overlying niche via adherens junctions containing a complex composed of Shotgun (Shg) and Armadillo (Arm) (Song and Xie, 2002). Adherens junctions between GSCs and the niche are not identical in the two sexes. At least one gene (gef26) regulates niche E-cadherin function in males but not in females (Wang et al., 2006). Loss of shg or arm function in GSCs results in a loss of stem cells from the ovarian niche indicating a loss of GSC anchoring (Song and Xie, 2002). Thus, adherens junctions are required to anchor GSCs to their niches in both sexes and this anchoring is necessary for GSC maintenance and self-renewal (Song and Xie, 2002).
Further, direct coupling of cap cells and GSCs via gap junctions is also important. Zpg is localized in the germarium at the interface between germ cells and somatic cells including GSCs and cap cells and GSCs and escort cells. Loss-of-function zpg mutant ovaries contain few GSCs and/or cystoblasts (Gilboa et al., 2003). In contrast to females, Zpg is not detected at the interface between GSCs and SSCs or between GSCs and the hub in males, indicating that the composition of gap junctions between GSCs and the overlying niche is not the same in males and females (Tazuke et al., 2002). Further it has been shown that different gap-junctional proteins allow different small molecules to pass through (Stebbings et al., 2000). This suggests that traffic between somatic and germ cells in the sexes could differ. Thus, while both adherence to the niche and gap-junctional intercellular communication are required for GSC self-renewal and for progression to mitotic stages in the two sexes, there are differences in the composition of both.
2.4. Signaling pathways
The same pathways utilized in the testis apex are deployed in the germarium, but not always to the same effect.
Jak/STAT signaling is required in both the testis apex and the germarium, but STAT activation is required in the germline of males as opposed to the soma of females (Fig. 4.4B). STAT activity is directly required within male GSCs where it confers stem cell identity. In contrast, the absence of Jak (due to loss-of-function in hop) in female GSCs is inconsequential (Gilboa and Lehmann, 2004). Nevertheless, loss of STAT function (through temperature sensitive alleles) results in drastically reduced GSC and escort stem cell numbers while increased Jak/STAT signaling, through overexpression of upd in germarial somatic cells, results in elevated numbers of cystoblasts and early cysts (Decotto and Spradling, 2005). STAT mutant germline clones do not affect normal development of germ cells but mutant ESCs and escort cells affect the development of closely associated germ cells (Decotto and Spradling, 2005). These germaria lack normal patterns of developing germline cysts and contain fewer GSCs with mislocalized spectrosomes (Decotto and Spradling, 2005). This suggests that female GSCs, cystoblasts, and cystocytes require a signal from the escort stem cells, not Jak/STAT per se. Activated STAT is thus required in escort stem cells, which in turn influence the female GSCs and affect their self-renewal.
TGF-β signaling is required in both male and female germ cells and has a much more conserved function. In both sexes, loss of TGF-β signaling leads to failed GSC self-renewal due at least in part to precocious derepression of bam expression. In both sexes, bam is also required for the cessation of mitotic divisions at the 16-cell stage and for progression to the next stage of differentiation. In females as in males, Dpp and Gbb ligands function synergistically to maintain GSCs by directly repressing transcription of the bam gene in GSCs (Fig. 4.4B and C) (Song et al., 2004; Xie and Spradling, 1998, 2000). dpp is transcribed at low levels in cap cells and escort cells and at higher levels in prefollicle cells, which are located more posteriorly in the germarium (Xie and Spradling, 2000). gbb expression is detected more generally in the soma including escort cells and early follicle cells and it may also be expressed in the terminal filament cells and cap cells (Song et al., 2004). The transcription pattern of these genes suggests that GSCs and cystoblasts are exposed to equivalent amounts of Dpp protein however, unlike in males where bam is repressed in both GSCs and gonialblasts; in females bam is repressed only in GSCs (Xie and Spradling, 2000). Therefore, intracellular modulation of sensitivity to Dpp signal, relieving Dpp-dependent transcriptional repression of bam, appears to be more important in the female germline (Casanueva and Ferguson, 2004).
In both sexes, the EGFR pathway is required for encapsulation of germ cells by somatic cells, which might be a prerequisite for proper interactions and reciprocal signaling between the two cell types (Fig. 4.4B). The somatic stem cells that are maintained in the male and female niches, interspersed between the GSCs are morphologically similar and produce nondividing daughter cells (cyst cells in males and escort cells in females), which enclose gonialblasts or cystoblasts and their progeny respectively. Male and female mutants lacking stet function show a similar phenotype: somatic cells fail to enclose germ cells and germ cells accumulate at early stages of differentiation. In females the accumulating cells resemble stem cells and cystoblasts, but in males the cells progress to the spermatogonial stage and mitotic interconnected cells accumulate indicating that EGFR signaling is required at a later step of differentiation in males than in females.
A comparison of the signaling pathways employed by the two sexes in their respective niches is outlined in Table 4.1.
Table 4.1.
Comparison of signaling pathways between male and female germ cell niche
| Male | Female | |
|---|---|---|
| Spectrosome positioning | Spectrosome is unanchored and not localized with respect to the hub | Spectrosome is always anchored at one pole of the spindle. Zpg-containing gap junction is present at the interface of the GSC and cap cell and is associated with the spectrosome, likely anchoring it |
| Establishment of spindle orientation | Spindle is oriented by centrosome position and anchored to the interface between GSCs and the hub by a complex consisting of APC2, Arm and Shg | Spindle is oriented by the spectrosome, which is anchored at the interface between the GSC and cap cell |
| Gap junctions | GSCs do not express Zpg | Zpg-containing gap junction is present at the interface of the GSC and cap cell |
| Germ cells lacking zpg function undergo differentiation, with spermatogonial cells accumulating | In females lacking Zpg, differentiation is arrested at the cystoblast stage | |
| Zpg is needed for communication between somatic cyst cells and spermatogonia | Zpg is required at an earlier stage than in males | |
| JAK/STAT signaling | Primary pathway controlling GSC self-renewal in males. STAT activity is cell-autonomously required in male GSCs where it confers stem cell identity, allowing stem cell self-renewal | STAT activity is not required in female GSCs but is required in escort stem cells which in turn influence self-renewal of GSCs. Thus, the requirement is indirect |
| TGF-β/BMP signaling | Required to repress bam transcription in the GSCs | |
| Loss of gbb/dpp function leads to loss of GSCs as the cells differentiate but fail to self-renew | ||
| bam is repressed in GSCs, gonialblasts and 2-cell spermatogonia and is required for cessation of mitotic divisions and differentiation into primary spermatocytes | bam is repressed in GSCs but BamC is expressed in cystoblasts and mitotically-active cystocytes. Bam is required for differentiation from GSCs into cystoblasts | |
| bam– male germ cells progress further in differentiation compared to bam– female germ cells with spermatogonial cells accumulating | In bam– females differentiation is arrested at the cystoblast stage | |
| EGFR pathway | Male germ cells mutant for stet progress to the spermatogonial stage until mitotic interconnected cells accumulate. EGFR signaling is required at a later step of differentiation | Cells resembling stem cells and cystoblasts accumulate in female stet mutants indicating EGFR signaling is needed during early differentiation |
3. SEX DETERMINATION IN THE GERM CELLS
The sexual dimorphism in the gonads detailed above is a manifestation of the divergent sexual identity of cells within the gonads. The process of sex determination within the soma differs from that within the germline. Somatic sex determination is a cell-autonomous process, however, germline sex determination is not strictly cell-autonomous, incorporating not only information about the number of X chromosomes (germline karyotype) but also information about the sexual identity of the associated somatic cells.
3.1. Somatic sex determination
The sexual identity of the somatic gonad depends on the splicing autoregulation of Sex-lethal (Sxl) to produce Sxl protein in females, but not in males (Bell et al., 1991). In the soma, the decision to be female or male is established in the early embryo (during mitotic cycles 12–14) when Sxl transcription is initiated in XX embryos but not in XY (or XO) embryos (Salz et al., 1989). Sxl is required for female-specific splicing of the transformer (tra) pre-mRNA (McKeown et al., 1987). Tra and the constitutively produced Transformer-2 (Tra2) protein are splicing enhancers that dictate female specific splicing of the doublesex (dsx) pre-mRNA, leading to production of the female-specific DsxF protein isoform (Burtis and Baker, 1989; Inoue et al., 1990, 1992; Nagoshi et al., 1988; Sosnowski et al., 1989). In males, Sxl and Tra are absent and dsxM is produced by default splicing, leading to production of male-specific DsxM (Bell et al., 1988; Salz et al., 1989). For details of somatic sex determination see the earlier chapter.
DsxF and DsxM are transcription factors that regulate development of multiple sexually dimorphic somatic structures including elaboration of abdominal pigmentation, development of external genitalia, sex combs, and abdominal neuroblasts (Baker and Ridge, 1980; Baker et al., 1989). DsxM is strongly expressed in all the somatic cells closely associated with premeiotic germ cells in males—the hub cells, the SSCs and cyst cells (Hempel and Oliver, 2007). This observation suggests that the male soma may continue to actively instruct the adjacent germline either by secreting positive “male” signals or by repressing “female” signals. By analogy, a similar mechanism may be in place in the female gonad where DsxF may be expressed in the somatic cells closely associated with premeiotic germ cells.
3.2. Germline sex determination
Key female germline sex determination genes are expressed at higher levels in female GSCs, cystoblasts, and cystocytes relative to the corresponding male germ cells at the equivalent stages (Fig. 4.5). For example, the ovo gene has two germline-specific transcripts, ovo-A and ovo-B (derived from alternative promoters), which encode C2H2 transcription factors with the same DNA-binding specificity but with opposite effects on targets (Andrews et al., 1998, 2000). In females, ovo transcripts are expressed in all germ cells, however, in males the transcripts are restricted to the apical tip, which likely corresponds with GSC, gonialblasts, and spermatogonia (Andrews and Oliver, 2002). Although both transcripts are expressed at higher levels in females when compared to males, ovo-B expression is higher when compared to ovo-A in females. The expression levels of both ovo-A and ovo-B are increased by the number of X chromosomes, thus the germ cell karyotype is a cell-autonomous cue resulting in higher levels of expression in females (Andrews and Oliver, 2002). A female soma specifically enhances expression of ovo-B, which becomes apparent in genotypes where (by specifically manipulating the somatic sex determination pathway) the XY soma alone is transformed from male to female without affecting the germline (Andrews and Oliver, 2002; Bielinska et al., 2005). Ovo isoforms bind to their own promoters in an autoregulatory loop and act on the ovarian tumor (otu) promoter resulting in higher otu levels in female germ cells when compared to male germ cells (Andrews and Oliver, 2002; Bielinska et al., 2005; Lu et al., 1998; Sass et al., 1995; Steinhauer and Kalfayan, 1992; Van Buskirk and Schüpbach, 2002). This transcriptional regulation matches an “incoherent type 1 feed-forward circuit model” (Mangan and Alon, 2003) for the transcriptional regulation of otu consistent with basal levels of ovo-A and ovo-B being determined by the number of X chromosomes and an autoregulatory loop, while sensitivity to a positive input from a female soma on ovo-B expression results in an enhancement of otu transcription levels. Input from the soma also boosts otu expression independent of ovo because otu expression is higher in ovo mutant XY germ cells within a female soma than in wild-type testis (Hinson and Nagoshi, 1999). In addition, high levels of otu expression also require the stand still (stil) gene which is expressed robustly in all female germ cells (Sahut-Barnola and Pauli, 1999).
Figure 4.5.
Germline sex determination pathway. Complete germ cell differentiation requires the evaluation of germ cell karyotype and integration of signal(s) from the somatic gonad to determine germline sex. (A) In females, ovarian tumor (otu) is a central player in integrating these two inputs. The 2 germline-specific ovo transcripts are expressed at higher levels in XX germ cells (than in XY germ cells) and thus the ovo locus appears to assess the germ cell karyotype. The proteins encoded by ovo-A and ovo-B are autoregulatory with opposite effects on transcription of their own promoters and the otu promoter. Somatic signal(s) from the female soma, downstream of tra, have a mild enhancement of ovo-B transcription and a more pronounced enhancement on otu transcription. The standstill (stil) gene is also required for robust otu expression. Downstream of otu, Sxl is sex-specifically spliced to produce protein in female germ cells. Along with sans fille (snf) and female lethal d (fl(2)d), Sxl autoregulates its own splicing. Ribosomal binding protein 9(Rbp9) is upstream of Sxl splicing regulation although it is not known precisely where it functions in the pathway. The fused (fu) gene likely functions in the soma to regulate female germline sex determination. (B) In males, little is known about germline sex determination. Both ovo and otu are expressed at lower levels (compared to female germ cells) but have no function. By default, Sxl is spliced into a male-specific isoform that does not encode a functional protein. It is not known how an XY karyotype is assessed or what may be downstream of the somatic signal which influences male germline sex determination. (Italics font denotes transcripts, Roman font denotes protein, gray color indicates lower levels than corresponding black color)
Sxl is spliced into a female-specific protein-encoding transcript downstream of otu regulation. This is based upon two lines of evidence: partial rescue of ovo and otu mutants by Sxl alleles that constitutively produce the female-specific transcript and the inappropriate expression of male-specific Sxl transcripts in ovo and otu mutant ovaries (Nagoshi et al., 1995; Oliver and Pauli, 1998; Oliver et al., 1993; Pauli et al., 1993). Sxl protein is found throughout the cytosol in the GSCs, cystoblasts and earliest cystocytes; thereafter Sxl protein levels abruptly decrease and nuclear foci appear (Bopp et al., 1993). The cytoplasmic localization of Sxl in GSCs and cystoblasts suggests that it may predominantly be functioning as a translational repressor in these cells, although that would not preclude its acting on targets with its better-defined role as a splicing factor (Bopp et al., 1993). Two genes required for Sxl splicing and autoregulation in the soma also function in the female germline: sans fille (snf) and female lethal(2)d (fl(2)d) (Hager and Cline, 1997; Ortega, 2005).
There are two additional genes whose mutant phenotypes place them in the same category as the other germline sex determination loci but whose precise roles remain uncertain. RNA-binding protein 9 (Rbp9), which is allelic to female sterile of Bridges (fes or fs(2)B), may be involved in Sxl autoregulation or otherwise upstream of Sxl regulation since mutant ovaries exhibit male-specific splicing of Sxl (Kim-Ha et al., 1999)(deCuevas, personal communication). Analysis of the fused (fu) gene has been confounded by its requirement as a component of the Hedgehog signal transduction cascade, which regulates the envelopment of germline cysts by the follicle cell layer (Besse et al., 2002). The Hedgehog pathway is not involved in germline sex determination, however, fu mutant ovaries express male-specific Sxl transcripts (Narbonne-Reveau et al., 2006; Oliver et al., 1993). Since mitotic clones in neither the germline nor the soma (in the follicle stem cell lineage) completely recapitulate the fu mutant ovary phenotype which has been shown to be ovary-autonomous by transplantation experiments, fu may function in the nonmitotic cells of the ovarian niche (the terminal filament and cap cells) or the escort stem cell lineage (Narbonne-Reveau et al., 2006; Smith et al., 1965).
Both ovo and stil are necessary for transcriptional activation of otu in the male germline; however, mutations in any of these genes have no effect on the male germline. Sxl is transcribed in the male germline but is not spliced into a protein-producing form. Even ectopic expression of Sxl protein in the male germline has no effect on spermatogenesis (Hager and Cline, 1997). Thus, the male germline sex determination pathway is distinct from the female germline sex determination pathway. Male germline sex cannot simply be a default fate since XY cells in a female soma have distinct phenotypic consequences (discussed below) strongly indicating that somatic signals must be incorporated into the germline sex decision.
4. GERM CELLS IN A SEX-MISMATCHED SOMA
Germline sex and subsequent germ cell development is affected in germ cells within a sex-mismatched somatic environment. This observation highlights the function of the niche in influencing the sex of the germline and also the differences between the male and female niches. The developmental consequences of male germ cells within a female soma differ from those of female germ cells within a male soma. Male germ cells within a female soma overproliferate, generally without differentiation (Fig. 4.6). Female GSCs within a male soma are not maintained—initially germ cells begin proliferating and generate cells resembling spermatogonia, but are unable to complete differentiation to produce functional sperm. Ultimately the cells undergo cell death (Fig. 4.7).
Figure 4.6.
Aberrant development of male germ cells within a female soma. Graphic representing postulated fate of a male GSC in a mismatched female somatic environment. The end result of this mismatch is over-proliferation of early germ cells that develop up to the spermatocyte and nurse cell stage. The follicles contain nurse cells, spermatocytes and small cells. Many of these are single cells others are connected by ring canals in small clusters. It is not yet known if any of these cells are truly intersexual.
Figure 4.7.
Aberrant development of female germ cells within a male soma. Graphic representing postulated fate of a female GSC in a mismatched male somatic environment. Some signaling pathways are shown to be disrupted (Jak-STAT and Hh) although the roles of other pathways can not be discounted. Development of a female germline within a male soma results in loss of germ cells. The germ cells develop up to the spermatocyte or nurse cell stage and finally degenerate.
4.1. Male germ cells within a female soma
The phenotypic consequences of male germ cells within a female soma can be observed in “mosaic intersexes,” which can be generated by various techniques (described in Fig. 4.8). Some mosaic delineations are very clear such as XY or XO germ cells transplanted into XX hosts with or without an endogenous germline whereas chromosomal mosaics such as gynandromorphs and triploid intersexes are not as clearly delineated (Janzer and Steinmann-Zwicky, 2001; Laugé, 1966, 1969a; Laugé and King, 1979; Marsh and Wieschaus, 1978; Steinmann-Zwicky et al., 1989; Van Deusen, 1977). Since the somatic sex determination regulatory cascade is well defined and the genes tra, tra2, and dsx are dispensable in the germline, XY or XO adults with a soma transformed from male to female by genetic manipulation of these genes produce clearly delineated sexual mosaics where the soma is sexually transformed from male to female but the germline is unaffected, at least autonomously (Andrews and Oliver, 2002; Bielinska et al., 2005; Hinson et al., 1999; Nagoshi et al., 1995; Waterbury et al., 2000). Such an XO soma transformed from male to female can completely support production of eggs from XX germ cells in gynandromorphs (Evans and Cline, 2007). In flies bearing mutations in germline sex determination genes, within an unaffected female soma there is an XX germline transformed from female to male (Gollin and King, 1981; Granadino et al., 1992; Kim-Ha et al., 1999; Oliver et al., 1990; Pauli et al., 1993; Schüpbach, 1985). Strikingly, all of the above mosaics have very similar phenotypes; the resulting XX “male” germ cells are indistinguishable from XY or XO “male” germ cells within a female soma. The germ cells within these somatic females never fully differentiate into eggs, instead, they remain mitotically active and poorly differentiated. While specific differences between mutants for discrete loci are important, clear trends emerge when considering all of these “mosaic intersexes” as a single class. In an effort to integrate what is known about GSC regulation and early germ cell differentiation with the phenotypic observations of male germ cells within a female soma (and vice versa in the section below), we have focused on literature with clear representation of specific germ cell developmental stages.
Figure 4.8.
Intersexes can broadly be divided into either “true intersexes” or “mosaic intersexes”. A number of different genetic or experimental manipulations can be used to generate mosaic intersexes useful for studying germline sex determination.
Within the female germarial niche, both GSCs and cystoblasts of male germ cells within a female niche resemble wild-type female GSCs and cystoblasts in morphology and probably number. A germarium houses 2–3 GSCs while the apical tip of the testis, due to its larger size, houses 6–9 GSCs (compare schema in Figs. 4.1B and 4.3C). The large GSCs are recognizable in the most apical position beneath the terminal filament in camera lucida drawings from EM sections of otu and fu mutants and in triploid intersexes with gonads morphologically resembling ovaries (King et al., 1978; Koch et al., 1967; Laugé and King, 1979). Further, the GSC has a spectrosome localized to the apex of the cell abutting the niche in Rbp9 and fu mutants as well as in germ cells of XY flies with a soma transformed from male to female (Janzer and Steinmann-Zwicky, 2001; Kim-Ha et al., 1999; Narbonne-Reveau et al., 2006). Anchoring of the spectrosome in GSCs is female-specific and is associated with a gap junction suggesting a cap cell may direct the formation of a gap junction with a GSC regardless of sex. Thus, there is a potential for miscommunication between male germ cells and female somatic cells. A female niche directs the early cell biology of male GSCs. In a wild-type female GSC, the spectrosome orients the mitotic spindles, even in the absence of centrosomes, whereas a wild-type male GSC requires centrosomes for spindle orientation (Stevens et al., 2007; Yamashita et al., 2003); but, it has not yet been tested if centrosomes or spectrosomes are required for GSC mitotic spindle orientation in male germ cells within a female soma. Any potential defects resulting from spindle orientation do not appear to affect the asymmetric division of the male GSCs in a female niche. In EM sections of germaria from females lacking fu and Rbp9, cystoblasts are discernable as single large germ cells subapically apposed to the GSCs, as in wild-type females, the caveat being that wild-type female cystoblasts and male gonialblasts are morphlogically indistiguishable (Johnson and King, 1972; Koch et al., 1967). Cystoblasts of Rbp9 and fu mutants also show BamC expression which is consistent with either bam derepression in these “male germ cells” because they respond like female germ cells or because they respond like male germ cells but derepress bam prematurely due to weaker TGF-β signal from an ovarian versus testis niche (Janzer and Steinmann-Zwicky, 2001; Kim-Ha et al., 1999; Narbonne-Reveau et al., 2006). Based on germ cell size and immunostaining for spectrosomes, the number of GSCs and cystoblasts may be increased relative to wild-type females in fu and otu mutants (Koch et al., 1967; Narbonne-Reveau et al., 2006; Rodesch et al., 1997). Excessive GSC numbers might be a male germline characteristic, as there are more GSCs in the testis apex than in the germarium although such a difference in GSC number may be the result of the difference in size of the stromal niche (surface area of hub cells versus cap cells) to which the GSCs are attached. Protein traps with enriched expression in GSCs and cystoblasts are now available and could be used to resolve this issue (Buszczak et al., 2007). Thus, no difference has been demonstrated between wild-type female GSCs or cystoblasts and those from “mosaic intersexes” of male germ cells within a female soma.
There are clear differences in the mitotic cystocyte divisions between wild-type female germ cells and male germ cells within a female soma. Wild-type female or male germ cells undergo four rounds of synchronous mitotic divisions with incomplete cytokinesis to yield a 16-cell cyst, however, when male germ cells are within a female soma the mitotic divisions become asynchronous, cytokinesis is occasionally complete and mitosis does not cease after four cell cycles. Examination of colchicine-pretreated germaria show equal numbers of 2, 4, or 8 cell clusters of cells with mitotic figures, however, in Rbp9 mutants there were excessive numbers of single cells or 2 cell clusters of mitotic figures and clusters of odd numbers of mitotic cells (up to 11) indicating that the mitoses are not synchronous and that supernumerary divisions occur (Johnson and King, 1972). While single germ cells (GSCs and cystoblasts) are normally located within 15 μm of the terminal filament, in Rbp9 mutants single germ cells can be found within the germarium as far as 100 μm away from the terminal filament consistent with some mitotic divisions culminating in complete cytokinesis (Johnson and King, 1972). In wild-type female cystocytes, the fusome grows by the formation of a new fusome plug within each newly forming ring canal and migrates (along with the ring canal) to join the central fusome (originially inherited in the cystoblast from the asymmetric division of the GSC). The central fusome pulls the ring canals into a rosette cluster, the mitotic spindle orients orthogonally to the central fusome for the next mitotic division resulting in an invariant “maximal branching” pattern of cystocyte connections (Storto and King, 1989). In XX flies with a germline transformed from female to male (Rbp9 mutant), cystocyte branching patterns suggest there may be a defect in mitotic spindle orientation in dividing cystocytes (Johnson and King, 1972; Koch et al., 1967). The fusome, which is derived from the endoplasmic reticulum and connects a common ER between the cystocytes, likely serves to facilitate communication between cystocytes allowing synchronization of the cell divisions (Snapp et al., 2004). Loss of synchronized mitotic divisions, odd numbers of cystocytes, lack of rosette configuration, and irregular cystocyte connection are all observed in hts hu li tai shao (ht3) mutant ovaries in which the fusome does not form (Grieder et al., 2000; Lin and Spradling, 1995). The fusomes in XY flies with a soma transformed from male to female or in XX flies with a germline transformed from female to male (otu, Rbp9, or fu mutants) are often spherical or dumbell-shaped similar to those of 2-cell cysts, or they are larger and either unbranched or irregularly branched (Hinson et al., 1999; Kim-Ha et al., 1999; Lin et al., 1994; Narbonne-Reveau et al., 2006; Rodesch et al., 1997). The lack of synchronized mitotic divisions and irregular cystocyte connections may both be consequences of defects in fusome function when male germ cells are within a female soma.
Sexual dimorphisms in the fusome and ring canal may be useful in the analysis of male germ cells within a female soma. With the cessation of mitotic divisions in wild-type female cystocytes, the fusome begins to break down and centrosomes travel along the fusome into the oocyte helping to establish the oocyte-nurse cell polarity within the cyst (Grieder et al., 2000). At the same time, female germline-specific ring canal growth begins which includes the concurrent appearance on the inner rim of the ring canal of Hts-RC and F-actin (which is relocalized from the fusome). Hts-RC is encoded by a female germline-specific transcript (Petrella et al., 2007). In wild-type male spermatogonia and spermatocytes, the cell cycle synchrony is maintained by the fusome, which does not break down until after the meiotic divisions (Wilson, 2005). There are dire consequences if the fusome is not maintained in spermatogonia: in hts mutant testes (which lack a fusome) spermatocytes frequently have too few or too many centrosomes and are sterile due to meiotic spindle defects and resulting aneuploidy (Wilson, 2005). While F-actin is present on the female fusome, it requires strong fixation protocols for reliable detection unlike the male fusome where F-actin is a major component and is easily detected (Huynh, 2006). Ring canals in wild-type male germ cells lack F-actin and Hts-RC but incorporate three septins (Pnut, Sep1, Sep2) not present in wild-type female germ cells (Hime et al., 1996). We have already described how male germ cells within a female soma often have spherical or dumbell-shaped fusomes or larger fusomes that are either unbranched or have an irregular branching pattern; these fusomes persist in germ cells well past the region 2a/2b transition and are even found within follicles. In otu mutant germ cells (in contrast to wild-type female cystocytes), substantial levels of actin filaments were seen in all fusomes examined and sometimes ring canals lacking F-actin encircled the fusomes—a combination seen in wild-type male ring canals and fusomes (Rodesch et al., 1997). Hts-RC is found in a few otu mutant germ cells and EM studies show the presence of outer rim but lack of inner rim deposits on the ring canals of otu and Rbp9 mutants (Johnson and King, 1972; King et al., 1978; Rodesch et al., 1997). Ring canals of XX germ cells transformed from female to male resemble spermatogonial ring canals, however, the presence of septins on the ring canals of male germ cells within a female soma has not yet been explored. Since the female germline-specific breakdown of the fusome is associated with active centrosome redistribution and inappropriate loss of fusome function results in centrosome redistribution in male germ cells it might be informative to investigate centrosome dynamics in male germ cells within a female soma. The examination of fusomes and ring canals suggests that male germ cells within a female soma may be more like wild-type male germ cells than wild-type female germ cells.
There is an increase of somatic escort cells in germaria of females with an XX germline transformed from female to male. In wild-type germaria, mitotically active germline cysts are enveloped by a pair of somatic escort cells that travel with the germline cysts through regions 1 and 2a of the germarium and undergo apoptosis at the border of region 2b (Decotto and Spradling, 2005). It is likely that wild-type escort cell apoptosis occurs in response to either loss of a prosurvival signal or initiation of a proapoptotic signal from the postmitotic germline cyst since in bam mutants, in which germ cells are arrested prior to the cystoblast stage and remain mitotically active, escort cells are present throughout the ovariole (Decotto and Spradling, 2005). However, the long-range signaling which maintains the follicle stem cells within their nonstromal niches at the same location, or interaction with the follicle stem cell lineage could regulate escort cell apoptosis. The only “mosaic intersex” for which expression of escort cell markers has been reported is in Sxl mutant germaria where there are an increased number of escort cells, however, marker expression does not extend throughout the ovariole but stops some distance from the terminal filament (Decotto and Spradling, 2005). The increase in escort cell number may be related to the aberrant cell divisions observed in male germ cells within a female soma where sometimes cystocyte divisions go to completion. Perhaps escort cells proliferate to envelop germ cells that are no longer connected by cytoplasmic bridges, or alternately they persist in association with male germ cells within a female soma longer than with wild-type female germ cells; the male equivalent of escort cells, the somatic cyst cells, remain in association with wild-type male germ cells throughout spermatogenesis. Since the cyst cells in males (and presumably escort cells in females) express sex-specific Dsx isoforms, and are in close association with the germline cysts at a time when aberrant phenotypes are manifest, further investigations into the association of escort cells with male germ cells within a female soma are warranted.
Similar to wild-type females, follicles form in region 3 of the germarium in flies that have male germ cells within a female soma but the continued mitotic divisions of the germ cells result in the eponymous phenotype “ovarian tumor.” Follicles can be found with hundreds to thousands of small germ cells, many of which are single cells or are connected by ring canals in small clusters of 2 or 3 cells (although in older flies there may be clusters with as many as 24 cells) (Gollin and King, 1981; King et al., 1957, 1978). Within these follicles, mitotically active cells can be found either as single cells or as clusters of germ cells interconnected by a fusome (King et al., 1957; Narbonne-Reveau et al., 2006). There are patches of bam expressing cells in tumorous follicles of fu mutants: fusome-interconnected clusters with high expression of bam, germ cells with low expression of bam and single cells that lack bam expression and contain a dot fusome (Narbonne-Reveau et al., 2006). The dot fusome could be indicative of either a GSC or cystoblast-like cell. Expression of bam within a wild-type germarium can be used to distinguish GSCs from cystoblasts; however, since bam should not be expressed in follicles at all, it is not clear if in these tumorous follicles it is a reliable indicator of GSC versus cystoblast identity. Use of other markers or short-term clonal lineage analysis may be useful in determining if some cells within these tumors divide asymmetrically like GSCs.
Within the tumorous follicles, some male germ cells within a female soma appear to begin differentiation along a male-specific or female-specific pathway. Germ cells found within ovaries containing XY transplanted pole cells or follicles of otu, stil, Sxl, or ovo mutants (as well as ovo-otu or ovo-snf interaction mutants) or triploid intersexes can morphologically resemble primary spermatocytes in nuclear positioning with distinct nucleoli and polar mitochondrial clouds visible by phase contrast microscopy in gonad squashes (Cline, 1984; Laugé and King, 1979; Oliver et al., 1990; Pauli et al., 1993; Pennetta and Pauli, 1997; Steinmann-Zwicky et al., 1989). The dense polar cloud of mitochondria is particularly diagnostic of primary spermatocytes prior to formation of the mitochondrial derivative (which goes on to form an important part of the sperm midpiece and tail during spermiogenesis), which suggests these cells have begun a male-specific differentiation pathway. This is consistent with the observation that male-specific SxlM transcripts are present in mutant ovaries (Lee et al., 2000; Oliver et al., 1993). Germ cells can also be found with “pseudo nurse cell” morphology with polytene rather than polyploid chromosomes—a chromatin state transiently seen in nurse cells in stage 4 follicles (King, 1970; Oliver et al., 1990, 1993; Pennetta and Pauli, 1997). In pseudonurse cells, bam expression is not detected, nor are fusomes present (Narbonne-Reveau et al., 2006). This suggests other germ cells begin a female-specific differentiation pathway. Germ cells with either male primary spermatocyte or female pseudonurse cell morphologies can even be found within the same ovariole (Pennetta and Pauli, 1997). This mosaicism is also evident from several sex-specific enhancer traps, which generally show patches of staining thoughout the ovariole with either male- or female-specific markers (reviewed in Oliver, 2002). By these criteria, germ cells with either male or female characteristics are present, but without concurrently looking for both male- and female-specific features at a cellular resolution, it is not possible to know if any of these germ cells represent true intersexes (initiating both sex-specific differentiation pathways). A possible reason for the patches of sex-specific morphologies and patchy expression of either male- or female-specific markers may be that male germ cells within a female soma are near a threshold of some binary switch between male versus female differentiation.
If the phenotypes (e.g., asynchronous germline divisions and continued mitotic divisions away from the apical niche) seen when male germ cells are within a female soma are due to the sex-mismatch between germline and soma, then one would expect that simultaneously transforming the sex of both soma and germline from female to male should restore male germ cell development. Indeed, when otu or snf mutant XX germ cells are within a soma transformed from female to male, the germ cells no longer overproliferate (Andrews and Oliver, 2002). Thus the otu and snf mutant phenotype is dependent upon signaling from a female soma. Interestingly, the germ cells in this situation do not produce sperm like XO germ cells within a soma transformed from female to male but rather resemble wild-type XX germ cells within a male soma where the few germline cysts do not progress past primary spermatocyte development and eventually degenerate (see Section 4.2 for more discussion of this phenotypic class) (Andrews and Oliver, 2002; Seidel, 1963). Despite the male germ cell characteristics of XX germ cells of female germline mutants, it is clear that these XX “male” germ cells are not capable of successfully completing male germline differentiation.
While we do not know what molecules signal from the somatic gonad to the germ cells to influence germline sex determination, it is worth considering whether any of the known soma-to-germline signaling pathways that we have discussed could result in the phenotypes seen when male germ cells are within a female soma.
The TGF-β pathway signals from the niche to the GSCs to repress bam and allow GSC self-renewal. Since male GSCs are anchored to the cap cells they should receive TGF-β signals similar to wild-type female GSCs. If the level of TGF-β signaling were higher (perhaps due to male germ cells being inherently more sensitive to the ligands than female germ cells), one might expect bam repression in the cystoblast and earliest cystocyte divisions leading to excessive rounds of mitotic divisions without differentiation. In other words, an excessive proliferation of small, undifferentiated male germ cells within the gonad, a phenotype strikingly similar to that observed when male germ cells are within a female soma. However, bam transcription is not repressed in male cystoblasts. Intriguingly, in tumorous follicles of fu mutants a reporter for receipt of TGF-β signaling (dad-lacZ) is expressed in germ cells which do not express bam (or do so at low levels); in wild-type female germ cells this reporter is only expressed in GSCs and cystoblasts (Narbonne-Reveau et al., 2006). Perhaps, away from the niche, male germ cells within a female soma are sensitive to low levels of TGF-β signaling whereas wild-type female germ cells are not. Alternatively, perhaps gene misexpression in male germ cells within a female soma results in inappropriate activation of TGF-β targets and overproliferation.
The major role of the Jak-STAT signaling pathway in the wild-type male gonad is to confer GSC self-renewal within the niche, but the major role of Jak-STAT signaling within the wild-type female gonad is to signal among somatic cells to refine subpopulations of follicle cells as the germline cyst becomes enveloped by follicular layer (Hombria and Brown, 2002). Perhaps male germ cells within a female soma are more sensitive to Jak-STAT ligands present in the germarium and follicles than wild-type female germ cells. In this scenario a male germ cell response would be adoption of GSC self-renewal characteristics, even away from the niche. This could be consistent with the phenotypes seen when male germ cells are within a female soma.
4.2. Female germ cells within a male soma
When female germ cells develop within a male soma (techniques outlined in Fig. 4.8), the resulting phenotypes are all very similar suggesting that the underlying defects and therefore the signaling pathways affected are essentially the same. Germ cells are observed only in newly eclosed XX adults with a soma transformed from female to male (tra− or tra2− loss-of-function mutants or dsxD gain-of-function mutants). However, gonads of XX first and second instar larvae with a soma transformed from female to male (tra−) are filled with dividing spermatogonial cysts which are surrounded by somatic cells, characteristic of male germ cell development (Seidel, 1963). These larval spermatogonial cells are mitotically active and divide to form germline cysts of up to 16 cells, germline cysts with greater than 16 germ cells are never seen (Seidel, 1963). Most gonads (60–70%) of XX adults with a soma transformed from female to male (tra− and tra2−) are devoid of germ cells; however, gonads of adults that contain germ cells have been examined and offer vital clues about the behavior of XX germ cells within a male soma (Hinson and Nagoshi, 1999, 2002; Nöthiger et al., 1989; Seidel, 1963).
In XX adults with a soma transformed from female to male (tra−), the GSCs are not attached to the hub; the unattached GSCs start to differentiate leaving behind an empty apical tip (Seidel, 1963). This is similar to the phenotype observed in gonads of wild-type female flies with mutations in components of the TGF-β or Jak/STAT pathways which are required for GSC self-renewal (directly or indirectly) suggesting that these pathways may be disturbed. If female GSCs receive insufficient Dpp and Gbb signals from the male niche, Bam would be expressed too early i.e. in GSCs, resulting in a bam overexpression phenotype (loss of the GSC population due to precocious differentiation). Insufficient Jak/STAT signaling would also lead to drastically reduced GSC numbers because the male SSCs may not be able to activate the same signaling pathway as ESCs, which would normally signal to a wild-type female GSC and help maintain its self-renewing capacity. Although Jak/STAT and TGF-β pathways are active in the niches of both sexes, female GSCs might receive insufficient signals due to differences in the expression levels between the sexes or due to an abolished or impaired cell–cell communication between female GSCs and the male hub and between female GSCs and male cyst cells. Furthermore, female GSCs might not be able to anchor properly within the male niche if there were an incompatibility of cell adhesion molecules and consequently would not be able to receive high enough levels of Jak/STAT or TGF-β signals and as a result fail to self-renew.
Cell–cell communication between GSCs and the niche may be abolished or impaired when germ cells of one sex are in contact with somatic cells of the opposite sex because the proteins composing gap-junction channels between germ cells and somatic cells in one sex may be different from those in the other. Zpg, for instance, is not detected at the interface between male GSCs and hub cells whereas in females Zpg is detected in foci at the interface between GSCs and the cap cells (Tazuke et al., 2002). This indicates that Zpg is perhaps required only by female GSCs to form functional gap junctions with the overlying cap cells. The putative partner to Zpg is thought to be Innexin 2 (inx2); its transcripts have been detected in female somatic cells immediately adjacent to germ cells expressing Zpg (Stebbings et al., 2002). Of the eight innexins in the Drosophila genome, ogre (inx1), inx2, and inx3 have been detected in female follicle cells and in addition inx2 is also expressed at low levels in the germline (Stebbings et al., 2002); however, in the male niche instead of Zpg a different innexin may be expressed. Testis ESTs for several innexins have been identified including ogre, inx2, and inx5 suggesting that different combinations of innexins may be required for the formation of gap junctions between GSCs and the niche in one sex versus the other (Tazuke et al., 2002). Since gap junctions are formed by a pair of hemichannels, one from each cell opposed in the narrow intercellular gap between neighboring cell membranes, incompatibilities between subunits would affect the assembly of the cell–cell channel (Curtin et al., 2002). Studies have shown that fly innexins, in general, cannot functionally substitute for one another (Curtin et al., 2002; Stebbings et al., 2000). It has also been proposed that when different heteromeric complexes form gap junctions, it may allow different signaling molecules to pass through the gap-junction channel suggesting that gap junctions between male hub cells and female GSCs may not allow passage of the same molecules as between male hub cells and male GSCs (Panchin, 2005). Thus, female GSCs within a male soma may not be able to receive the correct signaling molecules or the right levels of signaling molecules from the male hub required for their self-renewal.
Another reason for the loss of female GSC self-renewing capacity might be the fact that the male niche does not express female sterile (1)Yb (yb) and only very low levels of hedgehog (hh), genes required for female GSC self-renewal (Compare Figs. 4.2 and 4.4). yb regulates both piwi and hh (King et al., 2001). The yb locus encodes a novel protein expressed specifically in the cap and terminal filament cells and loss-of-function mutants exhibit significantly reduced expression of Piwi in cap cells and somewhat reduced expression of Piwi in terminal filament cells (King et al., 2001). Like loss-of-function mutations in piwi, loss-of-function mutations in yb lead to a depletion of GSCs in females likely due to differentiation of GSCs without self-renewing divisions (King and Lin, 1999). Yb is required for hh expression in the female niche (Fig. 4.4) (Forbes et al., 1996a,b). hh is expressed strongly in terminal filament cells and cap cells and at much lower levels in escort cells and plays a role in maintenance of GSC numbers, although its role may be partially redundant with Piwi (Forbes et al., 1996a; King et al., 2001). Consequently, absence of yb and hh in the male hub might lead to loss of female GSC self-renewal. Thus, an empty apical niche may be caused by one or more of the following: failure of female GSCs to attach to a male niche, failure to form functional gap junctions between the male niche and female GSCs and/or insufficient levels of signaling ligands from the sex-transformed male niche to the XX germ cells.
Most germ cells at later stages of differentiation in testes with XX germ cells within a male soma resemble spermatogonia (Laugé, 1969b; Nöthiger et al., 1989; Seidel, 1963; Steinmann-Zwicky et al., 1989). Although identical in size, cystocytes can be distinguished from spermatogonia by the structure and positioning of the nucleolus—in cystocytes, the nucleolous abutts the nuclear membrane and is consistently dark, while it is centrally positioned in spermatogonia and is round with a dark periphery and a bright center (Seidel, 1963). This indicates that the male soma has a strong influence on the XX germ cells directing them to develop such that they are highly biased towards the male pathway. Spectrosomes that elongate into fusomes connecting cells within germline cysts are seen in the spermatogonia of XX adults with a soma transformed from female to male (tra−). Male and female flies show a difference in their fusomes—F-actin is a major component of the male fusome but is not easily detectable in the fusome of Drosophila ovaries (Hime et al., 1996). F-actin is readily detected in the branched fusomes of XX adults with a soma transformed from female to male (tra−) further suggesting that these germ cells are highly male-biased in their development (Hinson and Nagoshi, 1999).
A few adult gonads also contain cells resembling primary spermatocytes. These cells have polar nuclei, mitochondrial clouds, and crystals in their cytoplasm (Laugé, 1969b; Seidel, 1963). Crystals are formed when spermatocytes develop without a Y chromosome (Meyer et al., 1961). In male host embryos transplanted with pole cells, spermatocytes with crystals are observed indicating that they are of the genotype XX or XO. The formation of spermatocytes in XX animals with a soma transformed from female to male or in males that received XX germ cells by pole cell transplantation also suggests that XX germ cells within a male soma differentiate with great bias toward the male sex differentiation pathway. Interestingly, most spermatocytes in XX animals with a soma transformed from female to male do not increase significantly in size in contrast to wild-type male germ cell development wherein spermatocytes undergo a 20-fold increase in cell volume (Laugé, 1969b). Eventually these arrested spermatocytes degenerate, further indicating an inability to complete spermatogenesis (Laugé and King, 1979; Seidel, 1963).
Although most germ cells in gonads of XX animals with a soma transformed from female to male (loss-of-function tra− or tra2−, gain-of-function dsxD) appear to resemble male germline stages, in some gonads germ cell clusters that resemble female germline stages are observed. These cysts were identified as female because they possess ring canals that incorporate the female-specific Hts-RC (Hinson and Nagoshi, 1999). Pseudonurse cells of varying sizes are also observed, although none reaching the size of true nurse cells (King et al., 1968; Nöthiger et al., 1989; Oliver et al., 1993; Seidel, 1963). These pseudonurse cells are found in groups but never form a cyst of up to 16 cells like a spermatogonial cyst. The fact that mitotic divisions were never observed in these cells supports the assumption that these cells are nurse cells, as polytene cells cannot divide (Brown and King, 1962; Seidel, 1963). Further, female-specific Sxl protein is also detected in some germ cell clusters suggesting that these clusters have a female identity (Oliver et al., 1993). The caveat is the lack of double labeling experiments with male and female germ cell specific markers, which would indicate whether germ cells exclusively express markers of one or the other sex indicating mosaic intersex identity or alternately, whether they simultaneously express markers of both sexes indicating a true intersex identity.
There are two possible explanations for the presence of female germ cell stages in XX animals with a soma transformed from female to male. XX germ cells may have an inherent tendency towards female development. In XX animals with a soma transformed from female to male this tendency might be suppressed by signals from the male soma, which actively secretes “male” signals. Only cells that do not receive a certain level of this masculinizing signal would be able to “escape” the suppression of female development. Consistent with this hypothesis, spermatogenic cell types are found in the absence of female cell types, but the reverse is never true (Nöthiger et al., 1989). Alternately, XX germ cells in absence of a positive feminizing signal from the soma may follow the male developmental pathway by default. In either scenario, the strength or extent of somatic sexual transformation of XX animals with a soma transformed from female to male (tra−, tra2−, dsxD) would determine the extent of germline sex transformation. Consistent with this hypothesis is the fact that XX germ cells can differentiate into egg chambers and mature eggs if the sexual transformation is only partial as in true intersexes (XX dsx) (Hildreth, 1965); whereas a stronger somatic sexual transformation as in gynandromorphs with a soma transformed from female to male due to loss of tra function leads to germ cell differentiation highly biased towards the male pathway (Nöthiger et al., 1989; Seidel, 1963). Furthermore, in testis of males transplanted with female pole cells, germ cells that exclusively follow the male developmental pathway are found and in 10–20% of these cases even immotile sperm are produced (Steinmann-Zwicky et al., 1989). This bolsters the hypothesis that the strength or extent of the somatic sex transformation determines the extent of germline sex transformation. The reason for some germ cells in gonads of XX animals with a soma transformed from female to male not receiving a high enough threshold of “male” signal may be because they are not properly surrounded by somatic cyst cells. An examination for cyst cell markers would be helpful to examine if pseudonurse cells in XX animals with a soma transformed from female to male indeed lack contact with somatic cyst cells.
Taken together, the data show that the majority of XX GSC that develop within a male soma are unable to self-renew and therefore die or differentiate. The few GSCs that do differentiate develop such that they are highly biased towards the male germline differentiation pathway. In a few gonads of XX animals with a soma transformed from female to male, in addition to spermatogenic stages, germ cells resembling pseudonurse cells are observed indicating that some cells initiate female germ differentiation. However, it is still unclear if these female germ cells are an indicator of the gonads having a mixture of true male and female germ cells constituting a mosaic intersex germline, or whether germ cells in these gonads simultaneously follow both male and female germline developmental pathways indicating a true intersex identity.
5. CONCLUDING REMARKS
Three major events mark adult germ cell development: first, GSC self-renewal, second, mitotic proliferation of germline cysts until they reach the 16 cell stage, and final, differentiation of the 16 cell cohorts such that they undergo synchronous meiosis in males and differentiate into 15 nurse cells and one oocyte in females. Germ cells may integrate signals from the soma with intrinsic cues at each one of these steps to progress to the next stage of gametogenesis. It is not yet clear whether germline sex determination requires continuous inputs from the surrounding soma or whether it is determined at a specific time-point during development and is irreversible. Different stages of germ cell development are affected when male germ cells are within a female soma and vice versa. For instance, GSC self renewal is only affected if female germ cells are within a male soma, not if male germ cells are within a female soma. This may indicate that male germ cells are more tolerant or capable of buffering somatic signals to a greater extent than female germ cells. Ultimately, neither male nor female germ cells can progress through gametogenesis in a sex-mismatched somatic environment to successfully generate functional gametes.
Unraveling the integration of somatic signals with germline autonomous cues would not only disclose the cause of the aberrant germ cell phenotypes observed when germ cells develop in a sexually mismatched soma, but would also lead to a detailed understanding of the differences between male and female niches in general. The pathway of cell autonomous cues required for germline sex determination in females is well on its way to being completely understood. However, the same is not true for males. Male germ cells must necessarily generate cell autonomous cues because they are clearly not exclusively dependent on the soma to determine their sex, as is obvious by the consequences of male germ cells within a female soma. The obvious next step in understanding germline sex determination in Drosophila is therefore to screen for XY germ cell autonomous cues needed for sex determination.
Elucidating the behavior of germ cells within their niche is fundamental to understanding germ cell development in Drosophila and, by extension, the requirement for and behavior of stem cells within their native niches in general.
ACKNOWLEDGMENTS
We thank members of the Oliver lab, Mary Lilly, Elissa Lei, and Jurrien Dean for helpful comments. This research was supported by the Intramural Research Program of the NIH, NIDDK.
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